The gram-negative peptidoglycan, which lies subjacent to the outer membrane, is relatively thin and undecorated by surface proteins or carbohydrates. The cell wall binding domain (CBD) epitopes are usually carbohydrates or teichoic acids that are unique to a species, much like a bacterial fingerprint. The most extensively studied lysins in animal models are Cpl-1, an N-acetylmuramidase, and PAL, an N-acetylmuramoyl-Lalanine amidase, both of which are from phages that infect Streptococcus pneumoniae. PlyG demonstrates lytic activity on a variety of Bacillus anthracis strains as well as one Bacillus cereus strain. Importantly, the enzyme was shown to be effective in killing and detecting germinating spores in addition to vegetative cells. The spore coat that normally forms an impenetrable surface for lytic enzymes undergoes an increase in porosity following germination, allowing lysins access to the peptidoglycan. Phage therapy has additional advantages of being self-replicating, has over 100 years of historical use, has obtained some regulatory approval, and can target either gram-positive or gram-negative organisms. Lysin therapy, in contrast, is not self-replicating and at the moment requires additional techniques to show efficacy on gram-negative bacteria. Clearly, both phage therapy and lysin therapy represent reasonable alternatives for management of food-borne pathogens.

This chapter examines the involvement of members of the genus Streptococcus as probiotics or as candidates for replacement therapy. Some researchers have turned to the development of replacement therapy strategies using relatively harmless indigenous streptococci as oral and nasopharyngeal probiotics, since (it is reasoned) these should have greater colonization potential than lactobacilli and bifidobacteria for these target tissues. The emphasis of the chapter is largely on current knowledge of the contribution of dedicated interbacterial inhibitors, the bacteriocins and bacteriocin-like inhibitory substances (BLIS), to the efficacy of streptococci as potential probiotics. It lists some of the practical factors such as safety, stability, formulation, colonization efficacy, and health benefits that may need to be taken into consideration when evaluating oral streptococcal probiotics. Acid resistance and adhesion to intestinal mucosa are desirable characteristics for traditional probiotics. For streptococcal probiotics targeting the oral cavity, acid tolerance is not a critical factor. Chronic multispecies bacterial infections of the oral cavity (e.g., dental caries, periodontal disease, and halitosis) are endemic, expensive to treat, and recalcitrant to conventional preventative protocols. These infections appear typically to be caused by the collective actions of more than one organism—the microbial community producing damage that individual microorganisms are probably incapable of inflicting. Intestinal probiotics are widely accepted for microbial population replacement and recolonization of the gastrointestinal tract, and a variety of beneficial strains are now inexpensively provided for the consumer.

By the year 1800, 200 years ago, the smallpox vaccine had established the principle of preventing serious disease by active immunization. In the 1940s, these whole-cell vaccines were supplanted by the next generation of pneumococcal vaccines, which consisted of the purified capsular polysaccharides (PS) of the bacteria. As early as 1891, animal experiments showed that killed pneumococci elicited protective immunity to subsequent challenge with virulent bacteria. The influenza pandemic of 1918 to 1919, together with the First World War, created a situation conducive to a highly increased incidence of pneumococcal pneumonia. Researchers administered a trivalent vaccine to 12,519 men in training at the United States. The results of the trials with the killed whole-cell vaccines were considered very encouraging at the time. Even a 30% reduction in numbers of cases of lobar pneumonia was welcomed because there were no alternative prevention measures. The experimental research of Avery, Heidelberger, and Goebel in the 1920s formed the essential links between pneumococcal capsules, their serotype specificity, and the identity of the capsules with PS that could be isolated from the bacterial cultures by chemical means. Despite recommendations for routine of pneumococcal immunization at-risk populations, the uptake of the vaccine was relatively slow. Nearly 10 years after the licensure of PS vaccine in the United States, only 10% of persons for whom the vaccine was recommended had ever been immunized. Protection against pneumococcal bacteremia, provided by the 23-valent PS vaccine, wanes over a period of 3 to 5 years, particularly in older people.

This chapter describes existing animal models of colonization and reviews what these various models have taught us about pneumococcal carriage. The most widely studied model of pneumococcal colonization is the mouse model. Infant rat models of pneumococcal colonization have also been described in several studies. A buccal mucosa model was also developed to evaluate the impact of ambient temperatures on pneumococcal colonization. The major limitation of chinchilla model is the relative lack of chinchilla reagents, as well as the expense. Animal models have provided information about the contribution of specific bacterial components to colonization, the first step in the pathogenesis of all pneumococcal disease. Different laboratories have used different mouse strains, which often vary in susceptibility to pneumococcal colonization and disease. Murine models of pneumococcal colonization have helped enhance the understanding of acquired immunity to pneumococcal colonization. The infant rat model of intralitter spread was used to evaluate the impact of systemic anticapsular antibodies in the prevention of pneumococcal colonization. The administration of bacterial polysaccharide immune globulin (from hyperimmune sera obtained from adults immunized with pneumococcal, Haemophilus influenzae type b, and meningococcal pure-polysaccharide vaccines) to infant rats reduced the likelihood of pneumococcal colonization by about 50%. The development of purified-protein vaccines or whole-cell vaccines, however, faces several challenges, including the inherent difficulty of studying colonization in animals that normally do not carry Streptococcus pneumoniae in their respiratory trees.

This chapter summarizes the relative prevalences of the most common serotypes prior to and following the introduction of the heptavalent pneumococcal capsular polysaccharide vaccine (PCV-7). It provides thoughts about the selection of serotypes for future-generation conjugate vaccines. A remarkable feature of the global epidemiology of pneumococcal carriage is the consistency of the dominant carriage serotypes in very different environments and at different times. Invasive disease potential, or invasiveness, is a measure of the ability of pneumococci to progress from nasopharyngeal carriage to invasive disease in humans. It differs from virulence in that the latter is often used to describe the ability of a pathogen to cause disease in laboratory animals. The 11-valent formulation prevented vaccine-related otitis media and was also shown to elicit antibodies with functional immunogenicity (opsonophagocytic activity) against 6A comparable to that seen with PCV-7. The incidence of invasive pneumococcal disease (IPD) due to vaccine serotypes has decreased substantially since the introduction of PCV-7 in the United States, in vaccinated children as well as all other age groups, indicating that pneumococcal transmission was interrupted as a result of the reduction in carriage in the vaccinated pediatric population. For mucosal disease, otitis media and nonbacteremic pneumonia, it is less clear which serotypes it would be most valuable to add since there appear to be less clearcut differences in invasiveness among serotypes. The only certain way of preventing mucosal disease is to sterilize the nasopharynx with respect to pneumococci.

Most bacteria that cause invasive disease, especially those that cause bacteremia, are protected from innate host immunity because they express polysaccharides (PSs) on their cell surfaces. The bacterial capsular PSs are composed of thousands of carbohydrate repeat units resulting in polydisperse polymers that can have molecular masses into the millions of daltons. Multivalent pneumococcal conjugate vaccines present additional complexities with regard to their syntheses, as each serotype is chemically distinct, effectively requiring the optimization of the manufacture of seven or more individual vaccines. Various proteins and peptide molecules have been demonstrated, in preclinical studies, to be effective carriers for PSs and oligosaccharides, but only a small number of protein carriers have been investigated in humans. Surface-exposed proteins and toxins from human pathogenic bacteria have been used as carriers, as they contain one or more of the T-cell epitopes. In order to convert PSs into T-cell-dependent antigens, the protein must be chemically linked to the carbohydrate; that is, there must be covalent links between the two components. Protein solubility at the required pH, concentration, and temperature is an important determinant of the suitability of a protein for use in a particular conjugation scheme. The conjugation step is generally the slowest chemical step and risks damage to the components. Efforts should be made to improve conjugation efficiencies to levels at which the residual unconjugated components, especially free PSs, do not interfere with inductions of protective immune responses. The use of efficient, mild conjugation chemistry would allow for higher yields of vaccine.

Several investigational pneumococcal conjugate vaccines (PCVs) have been evaluated in phase II immunogenicity and reactogenicity studies with infants. PCVs prevent mucosal infections (acute otitis media [AOM] and colonization), and thus, some groups have also made efforts to characterize the mucosal immune response after vaccination in the hope of finding serological correlates of mucosal protection. High-avidity antibodies can have greater functional capacity than low-avidity antibodies, and the increase in avidity is regarded as a marker of the development of immunological memory. The South African follow-up study suggests that HIV-infected children would benefit from a booster immunization while non-HIV infected children may have persistent protection due to natural boosting via pneumococcal colonization or cross-reacting antigens. The geometric mean concentrations (GMCs) of antibodies against diphtheria toxoid were generally higher in the group given PCV7-CRM in studies with both wP (15)- and acellular pertussis protein (aP)-containing combinations. In a number of PCV7-CRM trials, the response to a primary series of doses of a diphtheria-tetanus-pertussis combination vaccine (DTP) alone has been compared to the response to DTP coadministered with PCV7-CRM. PCV11-D-T given to infants at 18 weeks of age was able to boost the diphtheria toxoid and TT responses of Filipino infants who had received a DTwP vaccine at 6, 10, and 14 weeks. In a study of PCV11-D-T, a formulation with aluminum hydroxide induced higher GMCs of antibodies, but differences were not statistically significant. In general, there were no significant differences in the avidities of antibodies.

This chapter discusses the possible immune measurements that could be used to develop a correlative model, the functional forms of statistical models that have been used, and the results of correlative models for invasive pneumococcal disease (IPD), acute otitis media (AOM), pneumonia, and colonization. The most common immune measurement that is used in the development of protective correlates is the enzyme-linked immunosorbent assay (ELISA) for immunoglobulin G (IgG) class anticapsular polysaccharide antibodies. Generalization to other polysaccharide-based conjugate vaccines regardless of the carrier protein(s) is most likely acceptable, but the models have uncertain validity for nonconjugate pneumococcal vaccines that are protein or polysaccharide based or for populations that differ in important ways from those studied in the clinical efficacy trials, such as infants infected with human immunodeficiency virus. The probability of acquisition as a function of IgG antibody concentration was modeled using logistic regression. Only vaccine serotypes 9V, 14, 19F, and 23F were modeled because of the limited number of acquisition events for the other serotypes. In a separate analysis, serotype 6A was modeled using the immune responses to 6B. The antibody levels needed to protect against IPD, AOM, and colonization apparently differ. Higher levels are required for protection against AOM and colonization than for protection against IPD.

This chapter reviews both the direct and indirect effects of the introduction of pneumococcal conjugate vaccine into routine use. At this time, available data on vaccine impact are primarily from the United States, reflecting the timing of vaccine introduction. Reports from several sources indicate that the routine use of pneumococcal conjugate vaccine had a profound effect on invasive pneumococcal disease (such as bacteremia, bacteremic pneumonia, and meningitis) in children and that the effect occurred quickly following introduction. The shortages of pneumococcal conjugate vaccine that occurred between 2001 and 2004 meant that many children received an abbreviated two-dose series, received the primary three-dose series without the fourth (booster) dose, or experienced delays in the scheduled administration of doses. Pneumococci are generally transmitted from persons carrying pneumococci in the nasopharynx to others who become carriers; a small percentage of the new carriers will go on to develop disease. The reduction in carriage of vaccine serotypes in children who have received pneumococcal conjugate vaccine means that fewer vaccine serotype pneumococci are circulating among families, in day care centers, and in the community. Conjugate vaccines as currently designed can protect against only a limited number of the 90 pneumococcal serotypes. Data on vaccine impact on carriage and disease following routine introduction have added a wealth of information on top of that learned from clinical trials.

This chapter provides a summary of issues critical to the development and application of pneumococcal vaccines. In the preantibiotic era, vaccination attempts utilized whole killed pneumococci injected parenterally. Although such vaccines were sometimes protective in humans, they were also highly reactogenic. These killed vaccines were mainly used to elicit antibody in animals for passive treatment of infected humans. In 1933 it was clearly demonstrated that antibody to type-specific capsular polysaccharides (PS) could be highly protective. However, it soon became apparent that different strains of Streptococcus pneumoniae each expressed one of many different PS. Most subsequent vaccine attempts focused on the use of mixtures of the isolated PSs to elicit protection. However, the inability of young children to make adequate responses to most soluble PS led to the development and licensing of an immunogenic PS-protein conjugate vaccine for children. The problem of poor vaccine immunogenicity in children is being addressed by conjugation of the PS to protein carriers, thereby converting the PS from T-cell-independent to T-cell-dependent antigens. These antigens include the pneumococcal surface protein PspA; autolysin (lytA), an enzyme on the pneumococcal cell wall; and pneumolysin, a cytoplasmic protein that is released when pneumococci are autolyzed.

All bacterial species and phyla are involved in the phenomenon of resistance to antibacterial agents, sometimes posing genuine therapeutic problems. The strategy of Enterobacteriaceae faced with the aggression represented by the oxyimino cephalosporins was the production of conventional enzymes commonly known as extendedbroad-spectrum β-lactamases (ESBLs). The prevalence of nosocomial infections varied from 5 to 17% for patients admitted to ICUs in the hospital setting. In a survey conducted in central Europe, all of the strains of Staphylococcus aureus were susceptible to mupirocin, except in the case of Italy, where 1.5% of methicillin-resistant S. aureus (MRSA) strains were resistant. In S. aureus, the main mechanism of resistance of quinupristin-dalfopristin is methylation of the 23S rRNA with cross-resistance with macrolides, lincosamides, and streptogramin B. In the inducible type of resistance, quinupristin remains active because it is not an inducer of methylase; if constitutive, quinuprisitin is inactive and the combination become bacteriostatic due to alteration of quinupristin-dalfopristin activity in vitro and in vivo. S. pneumoniae is one of the major pathogenic agents of community-acquired or nosocomial parenchymatous respiratory tract infections (RTIs). The frequency of mutation of Mycobacterium tuberculosis varies according to the different antituberculosis agents. Antibiotic resistance in M. tuberculosis has long been known, particularly with streptomycin and isoniazid. Primary resistance of M. tuberculosis to isoniazid is greater than 10% in some countries.

This chapter outlines the impact of antimicrobial resistance by describing the epidemiology of select antimicrobial-resistant pathogens and the difficulties associated with treatment of infections caused by these organisms. It discusses antimicrobial treatment options for infections caused by resistant enterococci, Staphylococcus aureus, Streptococcus pneumoniae, and Mycobacterium tuberculosis. The arrival of vancomycin-resistant enterococci (VRE) is often attributed to the overuse of vancomycin, which has increased about 100-fold in the last 20 years, predominantly to treat methicillin-resistant S. aureus (MRSA), enterococcal, and Clostridium difficile infections. The most important risk factors for VRE colonization and infection include severe underlying disease, extended hospital stay, and previous antimicrobial exposure. In the absence of literature supporting the use of one agent versus another, linezolid should be used as first line therapy for VRE infections, because it can cover both Enterococcus faecalis and E. faecium. No population or geographical region is immune or isolated from the risk of infection with the primary cause of tuberculosis (TB) M. tuberculosis. Vaccine candidates that have been developed so far include recombinant M. bovis Bacillus Calmette-Guérin; attenuated M. tuberculosis; subunit and pooled subunit vaccines; fusion polyproteins; and DNA vaccines.

This chapter discusses clinical syndromes caused by Streptococcus pneumoniae, categorizing them based on pathogenesis and immune response. The current understanding of hematogenous infection is based on an expanded understanding of events in which pneumococci “settle out in” or “seed” various body sites. The resulting infections include meningitis, primary peritonitis, septic arthritis, osteomyelitis, and soft tissue infection. Important in the pathogenesis of acute sinusitis is congestion of the mucosal membranes caused by allergy or viral infection; resulting obstruction at the osteomeatal complex prevents clearance of bacteria. Not surprisingly, the bacteriology of acute maxillary sinusitis is similar to that of otitis media, with S. pneumoniae and/or Haemophilus influenzae being isolated in the great majority of cases. The majority of patients with pneumococcal pneumonia do not have detectable bacteremia, and it is very uncommon for the laboratory to isolate pneumococci from sputum of a patient who does not have a clear clinical picture of pneumonia or acute purulent tracheobronchitis. Empyema, the most common complication of pneumococcal pneumonia in the preantibiotic era, occurred in about 5% of cases and remains the most common today, with an incidence of approximately 2%. S. pneumoniae, despite its somewhat limited array of tissue-damaging enzymes and toxins, remains a prominent cause of infection with a surprisingly broad array of manifestations.

This chapter describes the mechanisms and consequences for the pneumococcal population of the selective pressures imposed by antibiotics and vaccines. While inhibiting pneumococci can have effects on other species of upper respiratory tract (URT) bacteria, the chapter focuses on the selective effects of vaccines and antibiotics on pneumococci. The direct effects of antibiotics on S. pneumonia in the nasopharynx of the treated patient depend on the pharmacokinetics and pharmacodynamics of the agent. Streptococcus pneumoniae resistance to the beta-lactam antibiotic class evolved mainly by complex restructuring of the targets of the beta-lactams, the penicillin-binding proteins (PBPs). To summarize the effect of antibiotics on S. pneumoniae ecology, it is clear that antibiotic treatment in any community profoundly affects S. pneumoniae ecology. Furthermore, a significant reduction of carriage of antibiotic-resistant and multidrug resistant (MDR) S. pneumoniae in the younger siblings of the day care attendees as the result of vaccination of their older siblings was observed. The chapter emphasizes the effects of vaccines and antibiotics on the ecology of pneumococci taking place against a background of a genetically diverse population structured by direct and indirect ecological interactions between strains. The importance of understanding pneumococcal population structure has been appreciated since the earliest days. This understanding can be extended to reconcile the disparate results of clinical trials of antibiotics and vaccines, to evaluate the relative selective effect of different antibiotics for resistant strains, and to project the effects of vaccines and antibiotics on future patterns in the prevalence of serotypes and resistant strains.

Streptococcus pneumoniae is one of the most important bacterial causes of respiratory infection and invasive disease in children and adults. This chapter reviews how increased rates of antibiotic-resistant S. pneumoniae have influenced the morbidity and mortality associated with pneumococcal disease and to present optimal therapeutic approaches for management of these infections in children and adults. In an eight-center children’s hospital surveillance study of pneumococcal infections, Kaplan et al. reported the outcome of 100 children with S. pneumoniae bacteremia secondary to penicillin- and cephalosporin-nonsusceptible infections. Informative studies suggest that an immunocompetent child between the ages of 3 and 36 months with culture-proven pneumococcal bacteremia, without meningitis, due to a nonsusceptible isolate can be effectively treated with a parenteral broad-spectrum cephalosporin as an appropriate initial therapy. Prior to beginning this therapy, repeat blood cultures should be obtained to document persistent bacteremia. Clinical presentation, cerebrospinal fluid indices on admission, hospital course, morbidity rates, and mortality rates were similar for patients infected with penicillin- or ceftriaxone-susceptible versus -nonsusceptible organisms. The relatively small numbers of nonsusceptible isolates and the inclusion of vancomycin in the treatment regimen for the majority of the patients limited the power of this study to detect significant differences between the groups. Pneumococcal isolates tolerant to vancomycin have been reported in cases of meningitis associated with poor therapeutic responses. Whether the increased use of conjugate vaccines and the reduced rates of inappropriate antibiotic use will lead to decreased antibiotic resistance to the pneumococcus in the future remains to be determined.

The clinical relevance of antibiotic resistance in the treatment of pneumococcal pneumonia has recently been reviewed. This chapter updates the review and expands it to the consideration of other pneumococcal diseases such as meningitis, otitis media, sinusitis, exacerbations of chronic bronchitis, and the limited literature on other types of infection such as infections of the pleura and endocarditis. Pharmacodynamics predict that high doses of intravenous penicillin remain useful for the treatment of pneumococcal pneumonia up to MICs of 4 μg/ml. Bacteremic pneumonia caused by a resistant strain has been described following trimethoprim-sulfamethoxazole therapy in a child and an adult and following prophylaxis with this agent, suggesting that the MICs of the agent for resistant strains probably exceed the levels achievable by oral dosing. Double-tympanocentesis studies have clearly demonstrated the relevance of pharmacodynamic principles for the prediction of bacterial eradication from the middle ear. As antimicrobial penetration into the cerebrospinal fluid is limited by the blood-brain barrier, lower levels of resistance are associated with clinical failure, which has been shown to occur even with intermediately beta-lactam-resistant strains. Pharmacodynamic principles explain the clinical failures observed with the emergence of resistance in some classes of antibiotics and also explain the successful continued use of the more active drugs despite the emergence of resistance. They thus allow the development of rational guidelines for the treatment of infections caused by antibiotic-resistant pneumococci.

Bacteria exist within populations, whether it is the population of pneumococci in the nasopharynx of an individual child or the isolates circulating within a local community, within a country, or globally. Multilocus enzyme electrophoresis (MLEE) is such a technique, and it has provided key insights into the population biology of many bacterial pathogens. The relationships among major lineages could be explored using a tree constructed from the concatenated sequences of all seven MLST loci. Pneumococcal clones diversify relatively rapidly, due mainly to the substantial impact of homologous recombination, presumably mediated by genetic transformation, which results in small segments of the chromosome of a recipient pneumococcus being replaced with the corresponding region from a distinct strain. Multiply antibiotic-resistant isolates of Streptococcus pneumoniae cannot have existed prior to the introduction of antibiotics into medicine and are probably less than 30 years old, yet considerable variation in the allelic profiles of the major resistant clones is observed. The carried population is thus of primary interest to the population biologist, and disease isolates need to be considered in the context of carriage. Antibiotic resistance might be expected to have arisen in those clones that were commonly carried in the nasopharynges of children. Serotypes that are rarely encountered in developed countries appear to cause a substantial amount of disease in some developing countries.

The human nasopharynx is the principal ecological niche for the heterogeneous population of Streptococcus pneumoniae (the pneumococcus), which exists as 90 different capsular types or serotypes. The focus of this chapter is to understand the behavior of S. pneumoniae and the factors that affect it in its normal habitat, the human nasopharynx. It is important to clarify that studies which have explored risk factors for pneumococcal carriage have not made a distinction between acquisition and carriage; therefore, data from these studies reflect risk factors related to the prevalence of pneumococci in carriage. There are little direct epidemiological data which show that acquired immunity plays a role in modulating carriage of pneumococci. The increase and subsequent decrease in pneumococcal carriage between 0 and 4 years of age is consistent with acquired immunity playing a role in reducing carriage, but the only direct evidence for natural immunity playing a role in preventing acquisition and thereby reducing carriage comes from a recent human challenge study. The conjugate vaccines have a marked and reciprocal effect on the acquisition and carriage of nonvaccine serotypes. Two additional beneficial effects have been observed in the human population following administration of the conjugate vaccine. The first is a significant reduction in carriage of antibioticresistant pneumococci, which is perhaps not surprising given that the major antibiotic-resistant clones are mainly of vaccine serotypes; the second is a reduction in the acquisition and carriage of vaccine-associated pneumococci in unimmunized younger siblings of vaccine recipients.

Antimicrobial susceptibility testing (AST) can be performed by four basic methods: disk diffusion, broth dilution, agar dilution, and gradient diffusion. The authors recognize that susceptibility testing methods and panels are subject to periodic modifications and that new systems and panels are introduced into various global markets as an ongoing process. This chapter reviews recently published evaluations of currently used commercial systems. The automated and semiautomated systems discussed in the chapter have the capability of producing standardized or customized patient test reports generated by computer software packages that are referred to as data management systems (DMS). In the chapter, five current automated systems are discussed individually and evaluations (advantages and limitations) are presented as an overview of the current literature. The automated Sensititre ARIS system is a broth microdilution method utilizing a standard 96-microwell panel containing serial dilutions of dehydrated antimicrobial agents. Panel configurations are currently available for gram-positive and gram-negative bacteria in either a MIC-only format or a breakpoint format and are listed in the chapter. The chapter describes published evaluations of several commercial MIC methods for Staphylococcus pneumoniae, and reviews several commercial methods for the rapid detection of methicillin-resistant S. aureus (MRSA).